P602: DISTINCT IMMUNE-RESPONSE PROFILE OF RICHTER TRANSFORMATION: HIGH EXPRESSION OF IL-10, LAG-3 AND OTHER IMMUNE CHECKPOINT MOLECULES
Authors: Q. Chen, A. Behdad, S. Ma, M. Schipma, Y.-H. Chen
Journal: HemaSphere (2022)

Background: Richter transformation (RT) is an aggressive progression of chronic lymphocytic leukemia (CLL), often to diffuse large B cell lymphoma (DLBCL), with poor prognosis and limited response to standard therapy. Our prior studies have shown differences in immune checkpoint molecule expression between RT and CLL; in particular, expression of programmed cell death 1 (PD1) in lymphoma cells and its ligands (PDL1) in background immune cells. Significantly, we have shown high PD1 expression in RT predicts its clonal-relatedness to CLL. Aims: To investigate immune-response pathway dysregulation in RT by gene expression profiling. To discover immune pathways and molecules that may serve as targets for immune therapy. To discover biomarkers that may help identifying patients that will benefit from checkpoint inhibitor therapy. Methods: We studied 16 patients diagnosed with RT and 18 with CLL. We performed a 400-gene targeted NGS gene expression profiling on RT and CLL cases using the Oncomine immune-response assay from ThermoFisher. Data analysis (RNA-seq with STAR and DESeq2) was done by aligning quality-sufficient reads to the human genome and counting reads for each gene. Gene normalization and differential expression were then calculated, with adjusted p-values <0.05 determining statistically significant differentially expressed genes. Next, a pathway analysis was performed (Metascape) to identify significant pathways among the differentially expressed genes. We also performed PD1 and LAG3 immunohistochemical stains in all cases. Results: Gene profiling analysis revealed significant differences in expression levels of 89 immune-response genes between RT and CLL cases. The top upregulated genes in RT comparing to CLL included several checkpoint pathway genes: LAG-3, HAVCR2/TIM3, and CD274/PD-L1. Several immune modulation/immune suppressive genes were significantly upregulated, in particular IL-10 and TGFb1. Other top upregulated genes included those involved in innate immunity (S100A9, S100A8, IGSF6), and in cytokine signaling (CCR1, IL-1B, IL-18). In addition to immune-response genes, MYC, TP63 and several cell proliferation pathway genes were upregulated in RT. The top down-regulated genes in RT compared to CLL included CD79B (B-cell receptor signaling), CD160 (checkpoint pathway), KLF2 and CD40LG (T-cell regulation), CIITA (IFNg signaling) and SELL (cell adhesion). To further confirm these findings we performed LAG3 IHC. LAG3 was highly expressed by large neoplastic B-cell in PD1-positive RT and only in scattered large cells in proliferation centers of CLL, and largely negative in PD1-negative RT cases. Average positive neoplastic cells were 67% in PD1+ RT (n=12), 2.8% in PD1- RT (n=4), and 10.8% in CLL (n=18) (P<0.05). Summary/Conclusion: Using a targeted NGS gene profiling analysis, we demonstrate a significant difference in expression levels of multiple immune-response genes in RT vs CLL. In particular, we found upregulation in immune modulation genes and checkpoint pathway genes PD1, LAG3 and TIM3, and immunosuppressive cytokine IL-10. Overexpression of LAG3 in large neoplastic B-cells of RT, specifically in cases clonally-related to CLL is an important finding that may serve as a diagnostic marker. Checkpoint molecules LAG3 and TIM3 can serve as new immune-therapy targets for the treatment of RT. IL-10 blockade has been shown to enhance T-cell immunity, and may be a potential therapeutic target.